The overall objectives of the proposal are to investigate the molecular mechanism of DNA replication and the evolution of genes essential for transformation in adenoviruses. The unique property of inverted terminal repetitions (ITR) common to all adenoviruses seems to play an unknown role in DNA replication. We have focused our effort in the past to determine the nucleotide sequences of this region from representative members of group A (highly oncogenic), group B (weakly oncogenic), group C and E (non-oncogenic) adenoviruses. The length and DNA sequence of this region diverge considerably except for a unique region of homology present at an exactly identical location near the termini. Analysis of the intermediates of replication of adenovirus types 2 & 5 from the injected cells or from the in vitro replication systems have shown, from the work cone in several laboratories, that initiation of DNA replication takes places at or near either end of the viral DNA. Displacement of one of the parental strands takes place as the daughter strand synthesis proceeds in the 5' to 3' direction. It is not clear how the displaced strand replicates. A model has been proposed that it replicates via a circular "panhandle" intermediate formed due to the presence of ITR. I propose to study the role of ITR in DNA replication of displaced parental strands using DNA cross-linking psoralens. The role of the unique 14 bp sequence which might function as a recognition signal for initiation of DNA replication will be studied by using chemically synthesized oligodeoxynucleotides as site-specific mutagens as well as by misincorporation of excisi-on-resistant analogues of deoxynucleoside triphosphates. Transitions, transversions and deletion mutants will be generated and assayed for DNA replication in vivo and in vitro. Our preliminary studies indicated that 9 amino acridine inhibits adenovirus DNA replication in vivo. We will pursue this aspect further to understand the molecular mechanism of this inhibition.